CN109061229A - A kind of scaling method of light field Micro-PIV system - Google Patents
A kind of scaling method of light field Micro-PIV system Download PDFInfo
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- CN109061229A CN109061229A CN201810876217.7A CN201810876217A CN109061229A CN 109061229 A CN109061229 A CN 109061229A CN 201810876217 A CN201810876217 A CN 201810876217A CN 109061229 A CN109061229 A CN 109061229A
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- G01P—MEASURING LINEAR OR ANGULAR SPEED, ACCELERATION, DECELERATION, OR SHOCK; INDICATING PRESENCE, ABSENCE, OR DIRECTION, OF MOVEMENT
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Abstract
The invention discloses a kind of scaling methods of light field Micro-PIV system, include the following steps: internal dense, the dilute calibration sample with trace particle of preparation；Utilize the distance of dense calibration sample combination definition algorithm calibration microlens array and microscope tube mirror；Utilize the distance in dilute calibration sample combination image similarity algorithm calibration sensor face and microlens array.The present invention replaces collimated white light source to demarcate using laser and fluorescence polystyrene microsphere, can eliminate bring error due to lambda1-wavelength difference, and stated accuracy is high.
Description
Technical field
The present invention relates to a kind of scaling methods of light field Micro-PIV system, belong to minute yardstick multiphase flow measurement technology neck
Domain.
Background technique
In recent years, microfluidic device, which is applied, becomes the hot spot studied both at home and abroad, the microfluidic device of different structure and function
With different flow behaviors.Studies have shown that since microfluidic device flow characteristics scale is small, active force between fluid molecule,
The surfaces such as electrostatic force stress effect is opposite to be enhanced, while being flowed also by microfluidic device configuration, wall roughness and wellability etc.
Factor influences, and flow behavior is extremely complex, and there is presently no suitable theoretical models to come to flowing complicated in microfluidic device
Characteristic explains.Flow visualization technique is the important experimental method of Characteristics of Micro Scale Flow research.Microscopic particles image is surveyed
Fast technology (Micro-scale particle image velocimetry, abbreviation Micro-PIV) may be implemented noiseless, whole
Field, transient state, quantitative minute yardstick velocity field measurement.Light field Micro-PIV system installs additional between microscope tube mirror and CCD camera
Microlens array, due to the presence of microlens array, light field Micro-PIV system single exposure can be obtained minute yardstick flow field
Field information further utilizes the three-dimensional information of trace particle in the available flow field of image processing techniques.Based on this feature,
Light field Micro-PIV system can realize the measurement of instantaneous 3D velocity field by one camera.
Light field Micro-PIV system requirements microlens array is positioned exactly at the imaging surface of microscope tube mirror, and CCD camera
Sensor cover be positioned exactly at the back focal plane of microlens array, at this point, system could farthest obtain the direction of light field
Information, while can be rebuild for subsequent deconvolution and accurate system parameter is provided.And in actual installation microlens array and CCD
When camera, it is difficult to guarantee its be in accurate location, especially the distance between microlens array and ccd sensor to be accurate to it is micro-
Meter level is other, this precision can not be realized by way of directly measuring distance.Therefore it needs using for light field Micro-PIV
The scaling method of system accurately installs microlens array and CCD camera.
Existing scaling method is chiefly used in macroscopical light field PIV system, only for the sensor cover and microlens array of CCD camera
Distance calibration, have ignored the calibration of the distance of microlens array and microscope tube mirror.This method using collimated white light source come
It is demarcated, collimated white light is converged by lenticule, and focal spot is formed on CCD camera sensor cover, is moved forward and backward CCD phase
Machine thinks that the sensor cover of CCD camera is located exactly at the back focal plane of microlens array when focal spot minimum.However it utilizes
When existing scaling method is demarcated, collimated white light source needs direct irradiation microlens array, and microscopical special construction, causes
Collimated white light source has to pass through object lens and cylinder mirror can just be irradiated to microlens array, and existing scaling method is not used to light field
Micro-PIV system.Therefore, still lack the scaling method for light field Micro-PIV system now.In addition, lambda1-wavelength
When different, the focal length of lenticule can generate variation, using the one-color fluorescence of wavelength 584nm when system practical operation, and white light wave
Long range is 380nm~780nm, therefore, when demarcating to light field Micro-PIV system, cannot use collimated white light source,
Otherwise error can be generated.
Summary of the invention
The technical problem to be solved by the present invention is to be directed to the missing of the scaling method of above-mentioned light field Micro-PIV system,
And provide a kind of scaling method of light field Micro-PIV system.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
A kind of scaling method of light field Micro-PIV system, which comprises the steps of:
Step 1: internal dense, the dilute calibration sample with trace particle of preparation；
Step 2: choosing dense calibration sample, place it on the objective table of fluorescence microscope, opens laser, passes through mesh
The dense calibration sample of sem observation, adjusts it with a distance from object lens, so that dense calibration sample is at micro objective focal plane；
Step 3: CCD camera is connected with relay system by bayonet, and CCD camera is in relay system back focal plane at this time,
CCD camera and relay system are moved integrally, it is adjusted at a distance from microlens array, before so that microlens array is in relay system
Focal plane；
Step 4: keeping the relative distance of microlens array, relay system and CCD camera constant, moves integrally camera system
System changes microlens array at a distance from microscope tube mirror, with dense calibration sample under CCD camera record different distance by micro-
The image formed after mirror and microlens array imaging；
Step 5: calculating the intelligibility evaluation value of image obtained by step 4, and when intelligibility evaluation value maximum, fixation is micro-
The position of lens array, microlens array is in microscope tube mirror imaging surface at this time；
Step 6: integral translation relay system and CCD camera change it at a distance from microlens array, until CCD camera
There is the tangent situation of adjacent sub-images in real-time interface, and the position coarse adjustment of CCD camera is completed at this time；
Step 7: choosing dilute calibration sample, place it on the objective table of fluorescence microscope, observes dilute calibration by eyepiece
Sample adjusts it with a distance from object lens, so that dilute calibration sample is at micro objective focal plane；
Step 8: the thin quasi- burnt spiral of rotation microscope records difference so that dilute calibration sample is in different depth from defocus
When depth from defocus, single trace particle is after microscope and microlens array imaging in the point of CCD camera imaging surface formation
Spread function (hereinafter referred to as PSF) image；
Step 9: calculate the resulting PSF image of step 8 under the same terms for being calculated by Abbe's theory of image formation
PSF image similarity function value；
Step 10: moving integrally relay system and CCD camera, finely tune it at a distance from microlens array, it is more different away from
From the lower image similarity functional value obtained by step 8 and nine, when image similarity functional value maximum, fixed relay system
System and CCD camera complete calibration.
In step 10, microlens array, relay system and CCD camera are coaxially fixed with cage bar using cage plate.
The calibration sample is prepared using fluorescence polystyrene microsphere, wherein the concentration of dense calibration sample is 20~25g/L；
The concentration of dilute calibration sample is 0.02~0.025g/L.
In the step 9, similarity function value is calculated using the image quality evaluation algorithm based on structural similarity
It arrives.
In the step 5, intelligibility evaluation value is calculated using the definition algorithm based on acutance.
In the step 8, different depth from defocus are respectively 40 μm, 50 μm, 60 μm, 70 μm, 80 μm and 90 μm.
Light field Micro-PIV system mainly by laser (λ=532nm), fluorescence microscope, camera system and calculates unit
At, wherein camera system is made of microlens array, relay system and CCD camera.CCD camera is mounted on rear side of relay system,
Relay system installed in front microlens array, camera system are connected entirely through interface with fluorescence microscope.The calibration mesh of system
Mainly have two o'clock: one is to ensure that microlens array is positioned exactly at the imaging surface of microscope tube mirror；Two are to ensure that CCD camera
Sensor cover is positioned exactly at the back focal plane of microlens array.At this point, system could farthest obtain the direction letter of light field
Breath, while can be rebuild for subsequent deconvolution and accurate system parameter is provided, to realize the measurement of instantaneous 3D velocity field.
When calibration, it is necessary first to the distance of microlens array and microscope tube mirror is demarcated, so that the accurate position of microlens array
At the imaging surface of microscope tube mirror, the sensor cover of CCD camera and the distance of microlens array are then demarcated, so that CCD phase
The sensor cover of machine is positioned exactly at the back focal plane of microlens array.If first demarcating the sensor cover and lenticule of CCD camera
The distance of array, then demarcate microlens array and microscope tube mirror apart from when, cameras record is sample by micro-
The image formed after lens array effect, is distorted image, and distorted image is not used to microlens array and microscope tube mirror
Distance calibration.Therefore, the calibration of two distances will follow the sequence of sensor cover after first lenticule.
Beneficial effect
Compared with existing scaling method, the present invention has the advantage that utilizing laser and fluorescence polystyrene microsphere generation
It is demarcated for collimated white light source, bring error due to lambda1-wavelength difference can be eliminated.Cooperate base using dense calibration sample
The calibration of microlens array and microscope tube mirror distance is completed in the definition algorithm of acutance, calibration is connected with clarity
System, stated accuracy are high；CCD phase is completed using dense, dilute image quality evaluation algorithm of the calibration sample cooperation based on structural similarity
Calibration is associated by the calibration of the distance of the sensor cover and microlens array of machine with image similarity, and stated accuracy is high.It is existing
Scaling method is demarcated based on focal spot, and collimated white light source needs direct irradiation microlens array, and microscopical special construction, is caused
Collimated white light source has to pass through object lens and cylinder mirror can just be irradiated to microlens array, therefore existing scaling method is not used to light field
Micro-PIV system, using scaling method of the present invention, final stated accuracy is up to 6 microns.
Detailed description of the invention
Fig. 1 system construction drawing；
Fig. 2 relative distance schematic diagram；
Fig. 3 intelligibility evaluation value figure；
Fig. 4 microlens array location position figure；
Fig. 5 relay lens and CCD camera location position figure；
Fig. 6 image similarity functional value figure；
Fig. 7 tests PSF and Abbe's theory of image formation PSF comparison diagram.
Wherein: 1- focal plane of lens, 2- micro objective, 3- convex lens, 4- beam expanding lens, 5- optical filter, 6- dichroic point
Light microscopic, 7- filter plate, 8- microscope tube mirror, 9- fluorescence microscope, 10 microlens arrays, 11- lenticule back focal plane, 12- relaying
System, 13- camera sensor face, 14-CCD camera, 15- laser, 16- computer, 17- cage bar, 18- cage plate.
Specific embodiment
With reference to the accompanying drawing, it elaborates to the present invention.
System construction drawing according to Fig. 1 assembles light field Micro-PIV system.Light field Micro-PIV system mainly by
Laser (λ=532nm), fluorescence microscope, camera system and computer composition, wherein camera system by microlens array, in
It is formed after system and CCD camera.CCD camera is mounted on rear side of relay system, relay system installed in front microlens array, camera
System is connected entirely through interface with fluorescence microscope.
As shown in Fig. 2, microscope tube mirror 8 uses L at a distance from microlens array 101It indicates, microlens array 10 and relaying
The preceding distance of camera lens L of system 112It indicates, the rear lens of relay system 11 use L at a distance from camera sensor face 123It indicates, mark
Fixed main purpose is to determine the size of these three distances.
The present invention is based on the scaling method of the light field Micro-PIV system of point spread function, specific steps include the following:
Step 1: dense, dilute calibration sample is made using fluorescence polystyrene microsphere.Wherein, the concentration of dense calibration sample is
20~25g/L；The concentration of dilute calibration sample is 0.02~0.025g/L.Particle is continuously distributed in dense calibration sample, dilute calibration sample
The discrete distribution of particle in product is being marked due to needing to use fluorescence polystyrene microsphere as trace particle when actual measurement
Also using the microballoon as calibration sample, this can guarantee the consistency of calibration with system parameter when actual measurement for timing；It is dense
Calibration sample can illuminate entire microlens array, so that the corresponding sensor cover pixel of microlens array has image
Formation, the accurate calibration of the distance for microlens array and microscope tube mirror and the sensor cover of CCD camera and lenticule
The coarse adjustment of the distance of array；Dilute calibration sample can form point spread function image, the sensor cover and lenticule for CCD camera
The accurate calibration of the distance of array；
Step 2: choosing dense calibration sample, places it on the objective table of fluorescence microscope 9, opens laser 15, passes through
Eyepiece observes dense calibration sample, adjusts it with a distance from micro objective 2, dense at this time until the sample observed is the most clear
Calibration sample is at the focal plane 1 of micro objective 2；
Step 3: opening camera software, is set as real-time interface, i.e., the image that computer 16 is shown is that CCD camera 14 is real
When the image that shoots, CCD camera 14 is connected with relay system 12 by bayonet, at this time the rear lens and camera of relay system 12
The distance L of sensor cover 133It is equal with relay lens focal length, so that camera sensor face 13 is in 12 back focal plane of relay system, L3Mark
It is fixed to complete, adjust the distance L of microlens array 10 and relay system 122, until the real-time interface display of camera goes out the most clearly
Lenticule grid, microlens array 10 is in 12 front focal plane of relay system at this time, i.e., microlens array 10 can be imaged on 1: 1
On camera sensor face 13；
Step 4: moving integrally microlens array 10, relay system 12 and CCD camera 14, fixes three's relative distance, i.e.,
Keep L2And L3Size is constant, changes microlens array 10 and 8 distance L of microscope tube mirror1, different L are recorded with CCD camera 141
The image that dense calibration sample is formed after fluorescence microscope 9 and microlens array 10 are imaged under value；
Step 5: calculating the intelligibility evaluation value of image obtained by step 4, and when intelligibility evaluation value maximum, fixation is micro-
The position of lens array 10, microlens array 10 is L on the imaging surface in microscope tube mirror 9 at this time1Calibration is completed.
The calculating of intelligibility evaluation value can be used based on Brenner gradient function, SMD (gray variance) function, energy ladder
Spend a variety of definition algorithms of function etc. and the definition algorithm based on acutance.Definition algorithm advantage based on acutance
It is relatively accurately reflect the clarity of image, calculates also very fast.It is selected in the present embodiment based on the clear of acutance
Clear degree algorithm.
The calculation formula of definition algorithm based on acutance is as follows:
Wherein: EAV indicates intelligibility evaluation value, and M and N are the line number and columns of image, and ∑ is summation sign, and i and a are tired
The cyclic variable put in marks in ∑, dI are grey scale change amplitude, distance increment of the dx between pixel.In practical applications, dI/dx
Desirable 8 neighborhood of pixel is calculated, and can use weight (distance increment) for horizontal and vertical direction is 1, and in 45 ° and 135 ° of sides
It can be set as 1/2 upwards.
Microlens array 10 and 9 distance L of microscope tube mirror when taking intelligibility evaluation value maximum1To demarcate apart from zero point,
Intelligibility evaluation value is with the variation diagram of calibration distance as shown in figure 3, the image that CCD camera 13 records when intelligibility evaluation value maximum
As shown in Figure 4.
Step 6: integral translation relay system 12 and CCD camera 14 change it at a distance from microlens array 10, that is, protect
Hold L1And L3Size it is constant, only change L2Size, until adjacent sub-images occur tangent at the real-time interface of CCD camera 14
Situation, as shown in figure 5, the position coarse adjustment of CCD camera 14 is completed at this time；
Step 7: choosing dilute calibration sample, places it on the objective table of fluorescence microscope 9, observes dilute mark by eyepiece
Random sample product adjust it with a distance from object lens 2, until the sample observed is the most clear, so that dilute calibration sample is in microscope
At 2 focal plane of object lens, the position of CCD camera 14 is further finely tuned；
Step 8: the thin quasi- burnt spiral of rotation fluorescence microscope 9, so that dilute calibration sample is in different depth from defocus, note
When depth from defocus is respectively 40 μm, 50 μm, 60 μm, 70 μm, 80 μm and 90 μm under recording, single trace particle passes through fluorescence microscope
9 and microlens array 10 be imaged after camera sensor face 13 formed image, this be test obtain PSF image；
Step 9: calculate the resulting PSF image of step 8 under the same terms for being calculated by Abbe's theory of image formation
PSF image similarity function value.Similarity function value can by image quality evaluation algorithm based on structural similarity,
PSNR and cosine similarity etc. are obtained.PSNR and cosine similarity these algorithms do not account for the structure of image, PSF image
Emphasis be exactly structure, so the present embodiment is preferably based on the image quality evaluation algorithm of structural similarity.
The calculation formula of image quality evaluation algorithm based on structural similarity is as follows:
Wherein: SSIM (T1, T2) it is image similarity functional value, T1, T2Two images respectively compared, μ1And μ2Respectively
It is T1And T2Average value, σ12It is T1And T2Covariance,It is T1Variance,It is T2Variance；c1=(k1L)2, c2=
(k2L)2It is for maintaining stable constant, L is the dynamic range of pixel value, k1=0.01, k2=0.03.
The PSF calculation formula of Abbe's theory of image formation is as follows:
Wherein: h (x, y, z) is PSF, x, y, and z is respectively point source coordinate, and j is imaginary unit, and ξ and η are respectively coordinate x and y
Spatial frequency；WithFourier transform and inverse fourier transform are respectively indicated, T (x, y) is the saturating of microlens array
Cross rate function, flIt is the focal length and wavelength of fluorescence of object lens with λ, U (v, u) is that point source is calculated by Abbe's theory of image formation in cylinder mirror
Imaging surface wavefront light distribution, expression formula are as follows:
Wherein: variable v and u indicate image surface laterally and axially optical coordinate, and M is the enlargement ratio of object lens, sin (α)=
NA, α are the half of objective aperture angle, and NA is the numerical aperture of object lens, and ρ is the normalization radial coordinate of objective aperture, and P (ρ) returns
One changes pupil function,J0() is zero Bessel function, x1, y1It is point spread function in image planes
On coordinate；
Step 10: moving integrally relay system 12 and CCD camera 14, finely tune it at a distance from microlens array 10, i.e., only
Change L2Size, more different L2The lower image similarity functional value obtained by step 8 and nine of value.Different depth from defocus
Image is one group, different distance (L2Value) it is different groups.The image similarity of the PSF for the particle that depth from defocus is 50 μm is with calibration
The variation diagram of distance is defined as the corresponding position of image similarity function maxima apart from zero point as shown in fig. 6, wherein demarcating, by
The variation of image similarity functional value can just be caused when only when the displacement of one pixel of the edge PSF generation, and the edge PSF is sent out
The corresponding L of displacement of a raw pixel2The variation of value, i.e. stated accuracy can obtain according to the following formula:
Wherein: Δ z is stated accuracy, pxFor pixel wide, Num is that microlens array transverse direction lenticule quantity and longitudinal direction are micro-
Small value in lens numbers, D are single lenticule aperture, fμlFor focal length of the single lenticule in a length of 584nm of incident light wave.
The device parameter of the present embodiment are as follows: px=5.5 μm, Num=66, D=136 μm, fμl=2260 μm, therefore the calibration of the present embodiment
Precision is 6 μm.
When image similarity functional value maximum, fixed relay system 12 and CCD camera 14 test PSF and Abbe at this time
Imaging theory PSF comparison diagram is as shown in fig. 7, image similarity functional value is as shown in table 1, and at this moment camera sensor face 13 records
Information is the light distribution of 10 back focal plane 11 of microlens array, i.e. the light distribution of the back focal plane 11 of microlens array 10 just may be used
With being imaged on camera sensor face 13 by relay system 12 1: 1, L2Calibration is completed；
1 image similarity functional value table of table
Step 11: closing laser 15, keeps microlens array 10, relay system 12 and CCD camera 14 coaxial, utilizes
Cage bar 17 is fixed with cage plate 18.
Calibration is completed.
Claims (6)
1. a kind of scaling method of light field Micro-PIV system, which comprises the steps of:
Step 1: internal dense, the dilute calibration sample with trace particle of preparation；
Step 2: choosing dense calibration sample, place it on the objective table of fluorescence microscope, opens laser, is seen by eyepiece
Dense calibration sample is examined, adjusts it with a distance from object lens, so that dense calibration sample is at micro objective focal plane；
Step 3: CCD camera is connected with relay system by bayonet, and CCD camera is in relay system back focal plane at this time, whole
Mobile CCD camera and relay system, adjust it at a distance from microlens array, microlens array are made to be in relay system front focal plane
Two
Step 4: keeping the relative distance of microlens array, relay system and CCD camera constant, moves integrally camera system, changes
Become microlens array with microscope tube mirror at a distance from, with CCD camera record different distance under dense calibration sample by microscope with
The image formed after microlens array imaging；
Step 5: calculating the intelligibility evaluation value of image obtained by step 4, when intelligibility evaluation value maximum, fixed lenticule battle array
The position of column, microlens array is in microscope tube mirror imaging surface at this time；
Step 6: integral translation relay system and CCD camera change it at a distance from microlens array, until CCD camera is real-time
There is the tangent situation of adjacent sub-images in interface, and the position coarse adjustment of CCD camera is completed at this time；
Step 7: choosing dilute calibration sample, place it on the objective table of fluorescence microscope, observes dilute calibration sample by eyepiece
Product adjust it with a distance from object lens, so that dilute calibration sample is at micro objective focal plane；
Step 8: the thin quasi- burnt spiral of rotation microscope records different defocus so that dilute calibration sample is in different depth from defocus
When depth, single trace particle is spread after microscope and microlens array imaging in the point that CCD camera imaging surface is formed
Function (hereinafter referred to as PSF) image；
Step 9: the calculating resulting PSF image of step 8 and the PSF under the same terms being calculated by Abbe's theory of image formation
The similarity function value of image；
Step 10: relay system and CCD camera are moved integrally, it is finely tuned at a distance from microlens array, compares under different distance
The image similarity functional value obtained by step 8 and nine, when image similarity functional value maximum, fixed relay system and
CCD camera completes calibration.
2. scaling method according to claim 1, which is characterized in that in step 10, using cage plate and cage bar to lenticule
Array, relay system and CCD camera are coaxially fixed.
3. scaling method according to claim 1, which is characterized in that the calibration sample uses fluorescence polystyrene microsphere
Preparation, wherein the concentration of dense calibration sample is 20~25g/L；The concentration of dilute calibration sample is 0.02~0.025g/L.
4. scaling method according to claim 1, which is characterized in that in the step 9, similarity function value utilizes base
It is calculated in the image quality evaluation algorithm of structural similarity.
5. scaling method according to claim 1, which is characterized in that in the step 5, intelligibility evaluation value uses base
It is calculated in the definition algorithm of acutance.
6. scaling method according to claim 1, which is characterized in that in the step 8, different depth from defocus are respectively
40 μm, 50 μm, 60 μm, 70 μm, 80 μm and 90 μm.
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| CN110174339A (en) * | 2019-05-28 | 2019-08-27 | 中国矿业大学 | A kind of multiple physical field coupling microscopic seepage experimental apparatus and method |
| CN110398480A (en) * | 2019-07-22 | 2019-11-01 | 中国科学院广州生物医药与健康研究院 | A kind of super-resolution microscope |
| CN111537765A (en) * | 2020-04-21 | 2020-08-14 | 东南大学 | Improved light field microscopic imaging device and construction method |
| CN113884414A (en) * | 2021-08-26 | 2022-01-04 | 湖南伊鸿健康科技有限公司 | Calibration counting plate and microscope lens calibration method |
| CN114153062A (en) * | 2021-12-07 | 2022-03-08 | 杭州纳境科技有限公司 | Super-surface objective lens, focusing method thereof and fluorescence microscope |
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